The goal of this proposal is to determine the role of tGLI1 (truncated glioma-associated oncogene homolog 1) in breast cancer brain metastasis (BCBM). Our laboratory discovered tGLI1 as an alternatively spliced GLI1 that lacks entire exon 3 and part of exon 4, but retains the ability to undergo nuclear transport and respond to sonic hedgehog-smoothened signaling. In addition to activating known GLI1 target genes, tGLI1 gains the ability to activate genes not regulated by GLI1, leading to increased migration, invasion and angiogenesis. Whether tGLI1 plays any role in metastasis of any tumor type is unknown. Our mouse studies indicated that tGLI1 promotes breast cancer preferential metastasis to the brain and conversely, tGLI1 knockdown selectively suppressed BCBM. tGLI1 protein is overexpressed in lymph node metastases and BCBM samples. In elucidating how tGLI1 promotes BCBM, we found that exosomes secreted from tGLI1-high cancer cells strongly activated astrocytes, the most abundant brain cells known to promote tumor growth when activated, and that tGLI1-high cancer cells had an increased ability to interact with and activate astrocytes in vitro and in vivo. Since microRNAs can be loaded into exosomes and circulating tumor exosomal miRNAs can prime distant organs for organ-specific metastasis, we conducted an exosomal miRNA microarray followed by validations and found that tGLI1-high cancer cells secreted high levels of exosomal miR-1290 and miR-1246 in vitro and in vivo. Whether miR-1290 and miR-1246 play any role in brain metastasis of any cancer or astrocytes is unknown. We observed that miR-1290/1246 inhibited expression of two transcription repressors (FOXA2 and SOX9), which leads to secretion of ciliary neurotropic factor (CNTF) to activate astrocytes and stimulate cancer cells. We hypothesize that tumoral tGLI1 promotes brain metastasis by priming astrocytes in the metastatic niche, and activated astrocytes in turn facilitate BCBM through secreting CNTF. We further hypothesize that tGLI1-high breast cancer cells prime astrocytes via the novel signaling axis: tumoral tGLI1?exosomal miR-1290/1246?astrocyte FOXA2/ SOX9?astrocyte CNTF?astrocyte activation. In Aim 1, we will determine the extent to which tGLI1 promotes BCBM using three mouse models (cell line-derived xenograft, PDX, and transgenic mice), overexpression and knockdown approaches, two inoculation routes (intracardiac and mammary fat pad injections), and human patient samples. In Aim 2, we will examine whether and how tumoral tGLI1 upregulates exosomal miR- 1290/1246 to activate astrocytes in the metastatic niche, and clarify how miR-1290/1246 suppress FOXA2 and SOX9 expression within astrocytes, how FOXA2 and SOX9 repress CNTF expression, and the role of CNTF in astrocyte activation and BCBM progression. In Aim 3, we will examine if miR-1290 and miR-1246 play essential roles in tGLI1-mediated BCBM, identify their downstream targets, and elucidate the roles of intracellular miR- 1290/1246 in BCBM. The project could define tGLI1, miR-1290/1246, and CNTF as novel mediators of BCBM and regulators of astrocytes in the metastatic niche, thus providing novel mechanistic insights into BCBM.